Intra-individual ITS polymorphism and hybridization in Pulmonaria obscura Dumort. and Pulmonaria angustifolia L. (Boraginaceae)

Internal transcribed spacer (ITS) is used as a molecular marker in most phylogenetical analyses in Boraginaceae, an unplaced group in the current angiosperm phylogeny. However, there is no knowledge on intra-individual polymorphism of ITS in Boraginaceae. Difficulties in PCR and sequencing of ITS in Pulmonaria species mentioned in the literature may be seen as indirect evidence of intra-individual polymorphism. This study aims to detect intra-individual polymorphism of ITS1–5.8S–ITS2 in rare species Pulmonaria angustifolia L. and common species Pulmonaria obscura Dumort. Cloning of ITS sequences of P. angustifolia , P. obscura and putative hybrid specimens from mixed population showed intra-individual polymorphism of ITS1–5.8S–ITS2 in all specimens, whereas most of them also contained pseudogenic sequences. Intra-individual sequence divergence of ITS1 and ITS2 spacers was similar in the P. angustifolia and mixed population and significantly higher than in P. obscura . P. angustifolia specimens had nucleotide polymorphisms characteristic to both P. angustifolia and P. obscura and part of P. angustifolia sequences clustered into mostly P. obscura clade on the neighbour-joining trees based on ITS1 sequence and ITS2 sequence and structure data. These results suggest a hybrid origin of all P. angustifolia populations studied. Thus, outbreeding depression is a likely explanation for the decline in populations near the northern border of distribution area of rare species P. angustifolia . Hybrid origin of the whole P. angustifolia is also probable; the central population from Poland showed a similar pattern of polymorphism as distribution edge populations.